Annotating all the sequenced genes in the human genome with functional information is a great challenge in the postgenomic era and requires animal models that allow powerful and high throughput forward and reverse genetic analysis of genes and gene clusters. The zebrafish (Danio rerio), a small fresh water fish, is especially suitable for this purpose. It combines the advantages of both invertebrates such as Drosophila (subject to large-scale mutagenesis and chemical screening) and mammals such as mouse (more similar to humans than the invertebrate models). A number of unique features of the zebrafish such as small size, external development, embryonic transparency, easy maintenance, and a large collection of genomic information make it an incredibly attractive model for functional genomics studies. However, this potential is not fully realized due to the lack of some critical genetic tools and resources in zebrafish. Gene targeting (knockout) by homologous recombination, one of the most powerful genetic tools utilized in the mouse model, is yet to be developed in zebrafish. Although alternative methods have been established in zebrafish, they do not produce true knockouts. Characterization of chromosomal rearrangement mutants created primarily by radiation has already demonstrated their great utility in studying gene function, genetic interaction of linked loci, and discovery of new genes. Inherited problems associated with these radiation-induced chromosomal rearrangements, such as difficulties in mapping DNA lesions and assigning functions to a particular gene when a deletion is large, have limited their wide application in the zebrafish community. Large collections of mutants with defined and molecularly marked chromosomal rearrangements that can be further manipulated are indispensable resources for the zebrafish community, for functional genomics, and for studying human disease. This application aims to develop a novel method for these needed resources. We integrate three well- established genetic tools in zebrafish into a two-transposon color reporter system for efficient gene targeting (knockout) and creating chromosomal rearrangements. Specifically, we utilize the high efficiency of Sleeping Beauty (SB) and Tol2 transposons for random chromosomal insertion and their ability to mobilize, the power of Cre/loxP system for site-specific recombination. Our use of fluorescent protein markers provides very high sensitivity for easy detection and selection of transposon movement and chromosomal rearrangements in the zebrafish. This strategy permits gene targeting (knockout) at pre-selected sites and provides a number of advantages over the existing irradiation and chemical mutagenesis methods. Several unique features of our methods and developed reagents make them applicable to individual zebrafish laboratories and can be scaled to genome levels. Mutants generated in this study will provide excellent resources for studying the function of these mutated genes in the zebrafish to better understand their role in humans and human diseases. PUBLIC HEALTH RELEVANCE: The zebrafish (Danio rerio) is a powerful model organism for studying human diseases. In this proposal, we will develop genetic methods for generation of zebrafish mutants with chromosomal rearrangements by using DNA transposons and the Cre-loxP technology. Ultimately, this work may lead to establishment of novel chromosome engineering technology and generation of mutant lines, which will provide excellent resources for scientists worldwide who wish to use this technology and study the function of these mutated genes in the zebrafish to better understand their role in humans and human diseases.